The objective of this research program continues to be elucidation of the role of proteolytic enzymes in normal lens development and cataractogenesis. Its focus is on the ATP-dependent (1500 kDa) and independent (700 kDa) forms of the lens multicatalytic proteinase complex (MPC). The first specific aim is to determine whether the active site mechanisms of the 700 kDa MPC are based on serine and/or cysteine residues and to identify which subunits are catalytic and which are regulatory. The investigators will use serine- and cysteine-directed radiolabeled inhibitors to identify the catalytIc subunits and molecular biology techniques to sequence selected subunits. Sequencing data will be used to determine relationships among the subunits of the lens MPC well as between the enzymes from different species and tissues. Using data from the Protein Sequence Data Base, they will compare the sequences with those of other proteinases whose catalytic mechanisms have been established. The second aim is to characterize the ATP-dependent proteolytic pathway in the lens. The 1500 kDa ATP-dependent form of the MPC will be assayed directly by hydrolysis of [125-I]-ubiquitin-labeled protein. The 1500 and kDa forms of the enzyme will be separated by native PAGE and identified by immunological techniques and by their abilities to hydrolyze chromogenic peptide substrates. Arg-tRNA:protein transferase, which catalyzes the first step in the ATP-dependent proteolytic pathway, will be characterized in the lens. Substrates of the arginylation reaction are also likely to be substrates of the ATP-dependent pathway and will be identified as [3H]-Arg-labeled proteins on 2-D gels. Oxidized crystallins will be tested as substrates for the ATP-independent pathway. Modulation of proteolysis in the lens, the third specific aim, will be determined by comparing and contrasting the levels of the ATP-dependent and independent forms of the MPC and the Arg-tRNA:protein transferase In lens tissues of different ages, species, and states of differentiation and pathology.